Oral administration of a non-absorbable plant cell-expressed recombinant anti-TNF fusion protein induces immunomodulatory effects and alleviates nonalcoholic steatohepatitis

Abstract

Aim: To evaluate the immunomodulatory effect of oral administration of PRX-106 in the high-fat diet model.

Methods: For 22 wk, C57BL/6 HFD-fed mice received daily oral treatments with BY-2 cells expressing recombinant anti-tumor necrosis factor alpha fusion protein (PRX-106). Mice were followed for serum liver enzyme and triglyceride levels, liver histology and intrahepatic and systemic FACS.

Results: The orally administered non-absorbable PRX-106 was biologically active. Altered distribution of CD4+CD25+FoxP3+ between the liver and spleen and an increase in the intrasplenic-to-intrahepatic CD4+CD25+FoxP3+ ratio and a decrease in the intrasplenic-to-intrahepatic CD8+CD25+FoxP3+ ratio were observed. An increase in intrahepatic NKT cells and a decrease in the intrasplenic-to-intrahepatic NKT ratio were noted. Assessment of the CD4-to-CD8 ratios showed sequestration of CD8+ lymphocytes in the liver. These effects were associated with a decrease in serum triglyceride levels, decrease in the aspartate aminotransferase levels, serum glucose levels, and HOMA-IR score. A decrease in hepatic triglycerides content was observed in the high dose-treated mice.

Conclusion: Orally administered PRX-106 shows biological activity and exerts an immunomodulatory effect, alleviating liver damage. The data suggest that PRX-106 may provide an oral immunotherapy for nonalcoholic steatohepatitis.

Keywords: Diabetes; Enbrel; Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis; Tumor necrosis alpha.

Figure 1

FACS analysis was performed on lymphocytes harvested from the spleens and livers of all mice from the experimental and control groups. The results were compared for the following subsets of cells. A: Intrahepatic CD4+CD25+FoxP3+ lymphocytes; B: Intrasplenic CD4+CD25+FoxP3+ lymphocytes; C: The intrasplenic-to-intrahepatic CD4+CD25+FoxP3+ ratio was calculated; D: Intrahepatic CD8+CD25+FoxP3+ lymphocytes; E: Intrasplenic CD8+CD25+FoxP3+ lymphocytes; F: The intrasplenic-to-intrahepatic CD8+CD25+FoxP3+ ratio was calculated; G: Intrahepatic NKT lymphocytes; H: Intrasplenic NKT lymphocytes; I: The intrasplenic-to-intrahepatic NKT ratio was calculated; J: To determine the effect of the treatment on the lymphocyte trapping in the liver, the CD4/CD8 lymphocyte ratio was calculated in the spleen and in the liver. The ratio between the splenic and hepatic CD4/CD8 ratios was calculated. ^aP < 0.05, ^bP < 0.01.

Figure 2

Serum levels of triglycerides (A) and aspartate aminotransferase (B) were measured in all mice from the experimental and control groups. AST: Aspartate aminotransferase. ^aP < 0.05.

Figure 3

A beneficial effect of oral PRX106 on fasting glucose levels was noted. Serum glucose levels were measured at the end of the study in all mice (A); the delta between the average serum insulin levels on day 0 and the end of trial was calculated (B); HOMA-IR was calculated for all mice at the end of the study (C).

Figure 4

The liver triglyceride content was measured and calculated per liver weight to determine the percentage of fat in the mouse livers. ^aP = 0.03.